Hepatitis C: Q80K polymorphism NS3 protease of hepatitis C virus genotype 1a; polymorphism D168A / H / T / V / Q of hepatitis C virus genotype 3; and resistance mutations antiviral drugs - RT-PCR and sequencing.
Infection with hepatitis C virus (HCV) infects the liver, causing in 50-85% of cases chronic infection, of which 2 to 20% progress to cirrhosis after 20 to 30 years of evolution and a small proportion of them made liver failure or hepatocellular carcinoma, and are candidates for liver transplantation. Many infected individuals are asymptomatic but infectious, so they can transmit the virus to new individuals.
The hepatitis C, has several well known genomic regions: 5'-NCR-C-E1-E2-p7-NS2-NS3-NS4A-NS4A-NS5A-NS5B-3'-NCR, encoding capsid (C ), envelope proteins (E1, E2), protease (NS3), cofactor protease, helicase and ATPase (NS4A), binding protein (NS4B), regulator of replication (NS5A), viral polymerase RNAP-RNAD (NS5B) . According to the nucleotide variability along its entire sequence differs in 6 genotypes when the differences are 30 to 35% of its nucleotide sequence. Each of the 6 genotypes subtypes can differ when a geotype differences in the nucleotide sequences of 20 to 25% of their sequence. Furthermore, within each subtype variants they can differentiate between them when differences of 5 to 8%.
Until recently, the only forms of treatment have been the ? interferon (PegIFN: pegylated interferon), associated or not with ribavirin. There are currently several groups of antiviral drugs, some already available and others under development. Some of these new drugs exert their effect by directly inhibiting some proper mechanism of virus replication, are called "direct inhibitors" of viral replication. These new drugs may be subdivided into a) Protease Inhibitors NS3 / 4A, that inhibit this protease necessary for (linear with ?-ketoamida replication: telaprevir, boceprevir; macrocyclic: Simeprevir, asunaprevir, vaniprevir, Danoprevir: linear tripeptide: faldaprevir; and other ABT-450r, GS-9256); b) inhibitors of the viral polymerase NS5B both nucleoside analogues (mericitabina, GS-7977, IDX-184) as non-nucleoside (filibuvir, GS-9190, BI-7127, ANA-598, VX-222 and ABT 333); c) non-nucleoside inhibitors of NS5A regulatory region (Daclatasvir).
Current therapy for chronic infection with hepatitis C virus genotype 1a, is based on the combination of an inhibitor of NS3 / 4A protease, together with peginterferon ? and ribavirin. This therapy is recommended for patients with compensated chronic infection, including cases that have evolved cirrhosis have not been treated or have failed therapy alone or associated to ribavirin.
Polymorphism Q80K and therapeutic response to protease inhibitors
Clinical trials with the new second generation inhibitors showed that efficacy is related to the absence of the polymorphism Q80K (change of amino acid glutamine for the amino acid lysine -Q- -K-) in the sequence of the protease encoded by the genomic region NS3 the virus.
Inhibitors of the NS3 / 4A protease above, the three most developed are: boceprevir (VictrelisR, Schering Corp / Merck & Co. Inc.), telaprevir (IncivekR, Vertex Pharmaceuticals Inc.) and second generation macrocyclic structure Simeprevir (OlysioR, Janssen Therapeutics). These protease inhibitors NS3 / 4A, are recommended for the treatment of infection by hepatitis C virus, genotype 1.
For antiviral drug efficacy, the best evaluation is performed through the sustained virological response (SVR: Sustained Virologic Response), ie it can not detect the virus, considering that the patient is cured.
The efficacy of therapy with protease inhibitors new hepatitis C, macrocyclic structure (e.gr. Simeprevir) is high (84%), when there exists in viral NS3 sequence polymorphism Q80K. Polymorphism means that in a given nucleotide sequence, a difference that involves an amino acid change in the protein sequence.
In U.S.A. it has been found that 48% of cases of infection with hepatitis C genotype 1a, has polymorphism Q80K, so the American FDA recommends a screening test ( "screening") is performed before initiating therapy inhibitors NS3 / 4A protease macrocyclic structure (eg Simeprevir), because when there is this polymorphism, efficacy reduces these drugs. In Europe the prevalence of this polymorphism Q80K, is lower (19%).
For the above reasons, polymorphism Q80K, is justified only determine in patients infected with hepatitis C virus genotype 1a, and when the virus has the amino acid change (substitution of glutamine -Q-, lysine -K-, in position 80), the NS3 / 4A protease, the therapeutic efficacy of protease inhibitors macrocyclic structure (e. g. Simeprevir) is reduced. However, the presence of this polymorphism does not affect the activity of protease inhibitors NS3 / 4A, with ?-ketoamida in structure (boceprevir or telaprevir e.gr.), or tripeptide structure lines (e.gr . falsaprevir).
In hepatitis C virus, genotype 3, there is D168A / H / T / V / Q polymorphism, which also reduces the activity of protease inhibitors NS3 / 4A macrocyclic structure (e.gr. Simeprevir).
Resistance mutations new antiviral drugs for chronic HCV infection
The high replication rate of HCV, along with the poor fidelity of its error correction polymerase (RNAdRNAp: HCV-RNA-dependent-RNA-Polymerase) generates a viral population with a diverse nucleotide sequence ( "quasispecies"). Differences in the nucleotide sequence involves substitution in the amino acid sequences may result in the lack of response to therapy with drugs that target the protein (e.gr. viral protease, viral polymerase, ...). Under the administration of an antiviral drug therapy selection is promoted, so that the resistant variant can become the majority viral population, leading to treatment failure. Hence the need to combine several drugs, such as the current recommendation to use pegIFN associated with ribavirin and an inhibitor of NS3 / 4A protease, to prevent the development of resistance.
In the case of protease inhibitors, several mutations conferring decreased or loss of sensation, some of them affecting most drugs (e.gr. V36A / M, T54A / V / S, R155T / known K, A156S / T / S / V, V170A / T / L), other affecting inhibitors possessing in its ?-ketoamida structure that forms a reversible covalent bond with the catalytic core of serine protease (e. g . boceprevir and telaprevir), and other mutations (D168V / a / T / H, A156S / V), involving linear tripeptide inhibitors that bind structure so noncovalent (e.gr. faldaprevir) and others affect the macrocyclic compounds (e.gr. Simeprevir).
Main known mutations:
Known mutations affecting protease inhibitors NS3 / 4A:
V36A / M, T54A / V / S, V55A, Q80K, R155T / K, A156T / S / V, D168V / A / T / H, V170A / T / L.
Mutations affecting non-nucleoside inhibitors of NS5A:
M28T, Q30H / R, L31M / F / V, P32L, Y93C / H / N.
Mutations affecting nucleoside NS5B polymerase inhibitors:
Mutations affecting non-nucleoside NS5B polymerase inhibitors:
C136Y / N, S365T / A, M414T / L, L419M / V, M423T / I / V, Y448C / H, I482L / V / T, M494I / A, P495S / L / A / T, P496A / S, V499A.
Tests in IVAMI:
• RT-PCR amplification and sequencing the NS3 region (Q80K polymorphism and mutations of resistance to protease inhibitors).
• RT-PCR amplification and sequencing of the NS5A region (resistance mutations NS5A inhibitors).
• RT-PCR amplification and sequencing of the NS5B (resistance mutations polymerase inhibitors) region.
• Plasma or serum, with viral load if possible ? 1000 IU / ml of HCV genotype 1a.
Inhibitors resistance mutations NS3 (protease), NS5A (NS5A inhibitors) or NS5B (polymerase inhibitors)
• Plasma or serum viral load if possible ? 1000 IU / ml of HCV.
Preservation and shipment of sample:
• Refrigerated (preferred, can be separated plasma or whole blood) for less than 2 days.
• Congelada (only if separate plasma from whole blood): more than 2 days.
Delivery of results:
2 to 4 days.
Cost of testing: